This study isolated two strains of Bacillus identified by cultural, morphological and biochemical methods as Bacillus subtilis (B1) & B. coagulans (B2). Potent antibacterial activity was observed against pathogenic and food-spoilage bacteria Staphylococcus aureus MTCC 1144, Pseudomonas aeruginosa MTCC 2488, Shigella flexneri MTCC 1457, Listeria monocytogenes MTCC 1463. MIC values of B1 and B2 were compared to those of standard strain B.subtilis MTCC 441 and potent activity was obtained at 640 AU of B1, 160 AU of B2 and 640AU of B. subtilis against S. flexneri MTCC 1457. Against S. aureus MTCC 1144, it was 320 AU of B1 and 640 AU of B2 and B.subtilis. Partially purified Anti-Microbial Substances (AMS) obtained from the two strains were low molecular weight polypeptides ranging from 5-25 kDa as detected by SDS-PAGE. Characterization of AMS by HPTLC, LC and GC-MS indicated presence of a mixture of secondary metabolites viz., derivatives of Benzenedicarboxylic acid, 3 OH Fatty acids like Hexadecanoic acid, Heptadecanoic acids and Quinolone compounds.  The findings of this study shed light on important aspects of food protection. Also due to the fact that Bacillus species have produced antibiotics in the soluble protein structure and that these antibiotics have been found to be cheaper and more effective in studies conducted to date, these microorganisms are preferable for commercial production.